Four main methods using peroxidase (HRPO) are now generally recognized for immunostaining. The methods are based on the immune reaction of an antigen to be detected in the specimen complexing with an antibody specific for the antigen. The methods differ primarily in the manner of detecting the antigen-antibody complex. The methods are the direct method, an indirect method using an enzyme-conjugated secondary antibody specific for the species of the primary or first antibody, the peroxidase-anti-peroxidase (PAP) method, and an indirect biotin-avidin method using a biotin-conjugated secondary antibody and a complex of a biotin-conjugated peroxidase and either avidin or strepavidin.
All of the immunohistochemistry methods, as well as other immunochemical methods, are multi-step procedures which consist of a sequence of reagent additions, incubations, and washings. Most of these procedures require highly trained personnel and the results can vary significantly between laboratories. Automated systems have been explored to introduce cost savings, uniformity of slide preparation, and reduction of procedural human errors.
For both automated and manual methods, there are a number of critical points to be considered. Care must be exercised to avoid the loss of specimen from the slide. Thorough washing of the specimen between reagent applications is essential particularly to remove unbound antibody as residues would be amplified. Excess liquid must be removed to avoid unwanted dilution of antibodies, yet specimens must never be allowed to dry out. Enough antibody reagent must be applied to completely cover the slide area where the specimen may occur, but waste has to be kept to an absolute minimum.
In addition, many of the reagents used in immunohistochemical methods as well as immunohistochemical methods, such as enzyme solutions and peroxidase color development reagents, have limited stability at the working temperature and even at room temperature. This necessitates frequent preparation of the reagents. Furthermore, nonspecific antibody binding, leading to erroneous results, remains a problem.
Methods and reagents that improve results and minimize reagent preparation would facilitate both manual and automated immunohistochemical methods. Many of the improvements could be readily applied to related immunochemical methods such as enzyme-linked immunosorbent assays (ELISA), immunofluorescence assays and in situ hybridization.